In patients with T2D, increased hepatic gluconeogenesis is the main cause of fasting hyperglycemia. Many ascribed increased gluconeogenesis to increased transcription of phosphoenolpyruvate carboxykinase (PEPCK). However, our recent findings challenge this dogma. We have shown that hyperglycemia and increased glucose production develop without increased expression of PEPCK or glucose 6-phosphatase in two rodent models with fasting hyperglycemia. We translated these results to humans, showing that in patients with poorly controlled T2D, fasting hyperglycemia developed without increased hepatic expression of PEPCK or G6Pc. Thus, another mechanism must account for increased gluconeogenesis in T2D. Our Preliminary Data now suggests that increased expression of pyruvate carboxylase may be this mechanism. We found that increases in PC protein occur in a rat model of T2D. Moreover, new human data shows that hepatic expression of PC mRNA variant-2 (PCV2) and PC protein, but not PEPCK or G6P, is tightly associated with HbA1c in non-diabetic subjects(R=0.80, P<0.001). The studies proposed in the Specific Aims of this grant will provide important new information on the role of PC in the pathogenesis of T2D. In addition, we will assess pyruvate carboxylase as a potential novel therapeutic target using a specific antisense oligonucleotide (PC ASO) to knockdown expression in liver and fat. In Aim 1, we will determine whether pyruvate carboxylase expression and activity is increased in patients with T2D. We hypothesize that fasting hyperglycemia in patients with T2D will be associated with increases in PCV2 mRNA, PC protein and PC activity. We will obtain liver samples from normoglycemic and patients with T2D undergoing elective abdominal surgery. This will safely provide a sufficient quantity of liver tissue to assess PC expression and activity, in addition to the expression/activity of other gluconeogenic enzymes and potential allosteric modifiers. We will relate the expression and activity of these enzymes to the fasting blood glucose concentration, and also pre-prandial glucose, mean blood glucose concentrations (using continuous glucose monitoring) and HbA1c. In Aim 2, we will determine how knockdown of pyruvate carboxylase affects basal and insulin stimulated hepatic glucose metabolism. Specifically, we will assess the compensatory pathways that may be activated using a sophisticated triple-tracer isotopic approach, direct quantification of key metabolite fluxes (e.g. glycerol and glutamine) and unbiased gene expression profiling. The efficacy of PC ASO in lowering endogenous glucose production and improving insulin sensitivity will be tested in two models of T2D, the ZDF rat and a transgenic rat overexpressing human islet amyloid polypeptide (HIP rat). In Aim 3, we will assess the effects of knockdown of hepatic and adipose pyruvate carboxylase on lipid metabolism. We show that knockdown of PEPCK in high-fat fed rats protects against adiposity but leads to nonalcoholic fatty liver disease and hepatic insulin resistance, akin to a mild lipodystrophy. In contrast, PC ASO also protects against adiposity but decreased liver fat and improved hepatic insulin sensitivity. We hypothesize that decreasing PC expression, but not PEPCK expression, will decrease hepatic glyceroneogenesis and, thus hepatic lipid storage. We will quantify hepatic and adipose glyceroneogenesis using sophisticated isotopic methods in fat-fed rats treated with PC ASO in comparison to PEPCK ASO and measuring differences in lipid metabolites (e.g. acyl-CoA's, diacylglycerol) by LC-MS/MS. We will also quantify changes in de novo lipogenesis in fructose-fed rats treated with either PEPCK ASO or PC ASO and assess the changes in NAFLD and hepatic insulin resistance. These studies will the first to quantify the effects of decreasing PC expression in vivo. In summary, the studies contained within this proposal could transform our understanding of the molecular regulation of hepatic gluconeogenesis in patients with T2D and validate pyruvate carboxylase as novel therapeutic target for both T2D and NAFLD.